Fluorescence recovery after photobleaching FRAP method

Noiiy Ring methods, pendant and spinning drop methods, for surface and interfacial dilational elasticity, thin-film techniques, and surface lateral diffusion when using fluorescence recovery after photobleaching (FRAP) methods. 

A very suitable method for measurement of the lateral diffusion of molecules adsorbed at the foam film surfaces is Fluorescence Recovery after Photobleaching (FRAP) ([491-496], see also Chapter 2). Measurements of the lateral diffusion in phospholipid microscopic foam films, including black foam films, are of particular interest as they provide an alternative model system for the study of molecular mobility in biological membranes in addition to phospholipid monolayers at the air/water interface, BLMs, single unilamellar vesicles, and multilamellar vesicles. 

In another approach, the interfacial diffusion of the nanoparticles was determined using two photobleaching methods fluorescence loss induced by photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP). It was found that the lateral diffusion of the nanoparticles at the interface as well as the diffusion normal to and from the interface deviated by about four orders of magnitude from the values obtained in free solution [46],... 

Despite the importance of interstitial transport parameters, it has been difficult to obtain accurate measurements of these values. Accumulation of molecules in tumor or normal tissue can be detected, but it is difficult to distinguish the roles of diffusion, convection, and binding, as well as transvascular transport. One experimental method that has been used successfully to quantitate interstitial diffusion, convection, and binding is fluorescence recovery after photobleaching (FRAP), in conjunction with tumors grown in transparent windows (Chary and Jain, 1987, 1989 Jain et al., 1990 Kaufman and Jain, 1990, 1991, 1992a, b Berk et al., 1993). 

One of the recent techniques most commonly used to measure diffusion in 2-dimensions is fluorescence recovery after photobleaching (FRAP). This method uses a laser beam focused through a fluorescence microscope to follow the diffusion of fluorescent molecules in a plane perpendicular to the laser beam. The fluorescence intensity from a laser spot of known diameter, typically a few microns, is measured. The laser intensity is then increased by approximately 1000 times. This irreversibly photobleaches any fluorophore in the spot. The intensity is then decreased again and the recovery in fluorescence intensity measured as unbleached molecules diffuse into the spot. The time function of fluorescence intensity is then analysed to give surface self-diffusion coefficient (Clark et al. 1990a, b, Wilde Clark 1993, Ladhaetal. 1994). 

Fluorescence recovery after photobleaching (FRAP) is a technique that allows the diffusion coefficients of a fluorescent probe to be measured. With a highly intense laser pulse all fluorophores in a selected spot are destroyed irreversibly, and the subsequent diffusion of fresh probe molecules into the area is followed by an increase in the fluorescence intensity. To give an example, gelatine-based organogels in microemulsions were investigated with this method [80]. 

Fluorescence recovery after photobleaching (FRAP) and the measurement of the fluorescence resonance energy (FRET), which allow the diffu-sional mobility of cellular components and their interaction at the molecular level to be monitored are other varieties of time-resolved methods [30]. A novel method is fluorescence correlation spectroscopy (FCS), which measures the statistical fluctuations of fluorescence intensity within a con-focally illuminated volume. Correlation analysis allows the concentration of particles and their diffusion to be determined [31], [32], FCS has proved... 

The nature of polymer motion in semidilute and concentrated solutions remains a major question of macromolecular science. Extant models describe polymer dynamics very differently 3-11). Many experimental methods have been used to study polymer dynamics (12). One meAod is probe diffusion, in which inferences about polymer dynamics are made by observing the motions of dilute mesoscopic probe particles diffusing in the polymer solution of interest. Probe diffusion can be observed by several experimental techniques, for example, quasi-elastic light scattering spectroscopy (QELSS), fluorescence recovery after photobleaching (FRAP), and forced Rayleigh scattering (FRS). 

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